Use of panaverium bromide for preventing cell proliferation and diseases caused thereby in the liver and digestive tract

ABSTRACT

The use of panaverium bromide for preparing a drug for treating or preventing diseases of the liver and digestive tract caused by excessive cell proliferation therem, is disclosed.

[0001] The present invention relates to a novel use of pinaveriumbromide in gastrointestinal pathologies, inflammatory digestive diseases(IBD), colites, ulcers, Crohn's disease or functional colopathies (IBS),during which proliferative phenomena which lead to pathologicalcomplications appear.

[0002] While the constant and rapid renewal of the epithelium of thedigestive tract is an important factor in the maintenance of theintegrity of the mucous membrane, it is also an important factor in thedevelopment of tumours and hypertrophies.

[0003] In these latter cases, an abnormal proliferation of theepithelial cells and an increased number of proliferative zones arealways observed; this is also found in patients who are at familial riskof cancer of the colon or in patients who are suffering from diseaseswhich predispose to cancer of the digestive tract (chronic gastrites,inflammatory digestive diseases, Crohn's disease, ulcerative colites,colonic polyps, etc.). Furthermore, it has been shown that inflammationof the intestine can give rise to hypertrophy and hyperplasia of thesmooth muscle cells at one and the same time. This results in athickening of the muscle wall, and therefore in a change in itsmotility, resulting in the appearance of complications which requiresurgical treatments.

[0004] In addition, inhibition of the proliferation of neoplastic cellsis a determining factor in the prevention and treatment of cancers andmetastases of the digestive tract.

[0005] Pinaverium bromide is the only calcium antagonist which isselective for the intestinal tract and which has spasmolytic activityand which is prescribed for the chronic treatment of functionaldigestive disorders (irritable bowel syndrome or IBS). This compound hasbeen described, in particular, in French patent 2 097 032.

[0006] The authors of the present invention have now surprisinglydemonstrated, by means of studies carried out in vitro, that pinaveriumbromide is able to inhibit the proliferation of neoplastic andhyperplastic cells of the digestive tract, with this inhibition beinggreater than that brought about by another calcium antagonist, i.e.verapamil. Furthermore, a study carried out in vivo also demonstratedthe efficacy of pinaverium bromide in decreasing tumours induced inanimals.

[0007] Pinaverium bromide can therefore be used as a medicament forpreventing cancer of the digestive tract both in the healthy individualand the individual suffering from a digestive pathology such as IBS. Inthe latter individual, it will then exert a prophylactic or therapeuticeffect which complements its effect on the IBS.

[0008] This compound may also be used for the therapeutic treatment ofhepatocarcinoma or of cancer of the pancreas and, more generally, of anycancer of the hepatodigestive tract.

[0009] The present invention therefore relates to the use of pinaveriumbromide for preparing a medicament which is intended for preventing ortreating diseases of the hepatodigestive tract which are due toexcessive proliferation of the cells of the tract.

[0010] More especially, the invention relates to the use of pinaveriumbromide for preparing a medicament which is intended for preventing ortreating intestinal cellular hyperplasias which are linked toinflammatory digestive diseases, or for preventing hypertrophies of thesmooth-muscle-cell wall of the intestinal tract and motor disturbanceswhich are due to the inflammatory diseases of the digestive tract(inflammatory bowel disease or IBD, Crohn's disease or ulcerativecolitis) which are characteristic of the IBS.

[0011] The invention is also directed towards using pinaverium bromidefor preparing a medicament which is intended for preventing or treatinga cancer of the hepatodigestive tract, comprising cancer of the colonand of the stomach, hepatocarcinoma and cancer of the pancreas, or elsethe carcinoid syndrome or functional diseases which are linked to thecarcinoid tumours.

[0012] It also relates to the use of pinaverium bromide for preparing amedicament which is intended for preventing and treating cancer of thehepatodigestive tract in patients who are suffering from irritablesyndromes of the colon (irritable bowel syndrome or IBS), whileeffecting a complementary and simultaneous treatment of the symptomswhich are characteristic of the IBS.

[0013] The invention also comprises a method for the prophylactic ortherapeutic treatment of diseases of the hepatodigestive tract which aredue to excessive proliferation of the cells of the tract, which methodconsists in administering a medicament containing pinaverium bromide, asthe active principle, to a patient.

[0014] Finally, the method is directed towards using pinaverium bromidefor producing a medicament which is intended for treating inflammatorydigestive diseases such as IBD (inflammatory intestinal disease orinflammatory bowel disease), Crohn's disease or ulcerative colitis,which diseases are generally associated with the thickening of themucous membrane.

[0015] Preferably, within the context of the present invention, thepinaverium bromide is used by being administered in a daily quantity ofbetween 50 mg and 450 mg. Administration is by the oral route or by theinjectable route, using a solution which is dosed at from 0.2 to 1mg/ml.

[0016] The advantages of the invention emerge in more detail in thetests which are described below.

[0017] In addition, the following figure shows the effect of pinaveriumbromide (▪) on inhibition of the growth of pancreatic carcinoid cells ascompared with the effect of verapamil (⋄).

[0018] Test 1:

[0019] Inhibition of the proliferation of colon cancer cells bypinaverium bromide as compared with the inhibition induced by anothercalcium inhibitor (verapamil)

[0020] The HT29 colon cancer cell line employed (originally isolatedfrom an adenocarcinoma of the human colon) was obtained from the ATCC(American Type Culture Collection).

[0021] The cells are cultured at the rate of 10⁴ or 5×10⁴ cells in awell containing 200 μl of culture medium composed of RPMI 1460supplemented with 10% fetal calf serum, 2 mM of L-glutamine and 25 μMgentamicin. Pinaverium bromide (or verapamil), dissolved in 50% ethanolor DMSO, is added to the cell culture at various concentrations, and thepercentage inhibition of cell proliferation is assessed by measuring theincorporation of tritiated thymidine (³HThdR), with the latter beingadded to each well at the rate of 37 KBq (1 μCi); the contents of thewells are withdrawn and counted 15 hours later using a “Filter Mate™Cell Harvester and Matrix 9600™ Direct Beta Counter” (Packard).

[0022] The results are as follows: Concentration of % Inhibition theproduct tested pinaverium % Inhibition (% by weight) bromide verapamil³HThdR 0.025 78 17 0.05 82 17 0.1 86 27

[0023] The inhibitory effect of the pinaverium bromide is from 3 to 5times greater than that of the verapamil.

[0024] Test 2:

[0025] Effect of pinaverium bromide on hepatocarcinoma cells.

[0026] LFC rat hepatocarcinoma cells (VPR42, Villejuif) are cultured atthe rate of 10⁴ or 5×10⁴ cells in a well containing 200 μl of culturemedium. The products to be tested (pinaverium bromide and verapamil) aredissolved in 50% ethanol and used at concentrations of 0.1% and 1% byweight.

[0027] Inhibition of cell proliferation is assessed using tritiatedthymidine as described in Test 1. The assessment is made in comparisonwith the reference product, which is verapamil. The results show apercentage inhibition of proliferation of from 85 to 88% in the case ofpinaverium bromide and of 72% in the case of verapamil.

[0028] Test 3:

[0029] Effect of Pinaverium Bromide on the Growth of STC-1 CarcinoidIntestinal Cells

[0030] The STC-1 mouse intestinal cancer cell lines are cultured in aDMEM medium which contains 15% horse serum and 2.5% FCS. The products tobe studied are added to the culture and cell growth is assessed bycounting cells or, more frequently, by a fluorescence technique which isbased on the cellular enzymic hydrolysis of the fluorogenic substrate4-methylumbelliferyl heptanoate (MUH); thus, viable cells which areincubated with MUH generate a fluorescent signal which is proportionalto their number and which is determined using a “Titentec Fluoroscan II”(Flow Laboratory), with the value being given in arbitrary fluorescenceunits.

[0031] Pinaverium bromide was found to be twice as active as verapamilin the test.

(ED₅₀ PINAVERIUM=7 μM ; ED₅₀ VERAPAMIL=13 μM)

[0032] Test 4:

[0033] Study of the Inhibition by Pinaverium Bromide of the Growth ofBON Pancreatic Carcinoid Cells

[0034] The BON human pancreatic carcinoid cell lines are cultured in aDMEM/F12K (1/1) medium to which 10% FCS is added. The verapamil and thepinaverium bromide are added to the culture and cell growth isdetermined after 8 days by means of a fluorescence technique such asthat described in Test 3.

[0035] The results reported in the figure demonstrate the efficacy ofpinaverium bromide in inhibiting the growth of the BON pancreaticcarcinoid cells, and its superiority to verapamil

(ED₅₀ PINAVERIUM=7.2 μM ; ED₅₀ VERAPAMIL=43.6 μM).

[0036] Test 5:

[0037] Inhibition by Pinaverium Bromide of the Hyperplasia of RatIntestinal Smooth Muscle Cells Which is Induced by PDGF(Platelet-derived Growth Factor)

[0038] The intestinal smooth muscle cells are cultured in a culturemedium containing 10% fetal bovine serum and then exposed to PDGF(growth factor found during inflammations) for 20 hours. Cell growth isassessed by the incorporation of tritiated thymidine.

[0039] Pinaverium bromide (10⁻⁵ M) totally inhibits the growth of theintestinal smooth muscle cells which is induced by the inflammationfactor PDGF. It therefore has a beneficial effect on inflammationphenomena of the digestive tract.

[0040] Test 6:

[0041] Effect of Pinaverium Bromide on the Diameters of the Tumourswhich are Induced in Nude Mice.

[0042] The tumours are induced by subcutaneously injecting HT29 coloncancer cells into nude mice.

[0043] Pinaverium bromide is injected intraperitoneally a few hoursbefore injecting the HT29 cells.

[0044] Tumour development is assessed by measuring the diameter of theinduced local tumour in relation to the duration of the experiment,which is 44 days.

[0045] A 30% reduction in the diameter of the tumours is observed fromthe 25th day of the experiment onwards.

1. Use of pinaverium bromide for preparing a medicament which isintended for preventing or treating diseases of the hepatodigestivetract which are due to excessive proliferation of the cells of thetract.
 2. Use according to claim 1, characterized in that the diseasesof the hepatodigestive tract are intestinal cellular hyperplasia orhypertrophy of the smooth-muscle-cell wall of the intestinal tract andmotility disturbances which are due to the inflammatory diseases of thedigestive tract (inflammatory bowel disease or IBD, Crohn's disease orulcerative colitis) which are characteristic of the irritable syndromesof the colon (IBS).
 3. Use according to claim 1 for preparing amedicament which is intended for preventing or treating a cancer of thehepatodigestive tract, comprising cancer of the colon and of thestomach, hepatocarcinoma and cancer of the pancreas, or else thecarcinoid syndrome or functional diseases which are linked to thecarcinoid tumours.
 4. Use according to claim 1 for preparing amedicament which is intended for preventing and treating cancer of thehepatodigestive tract in patients who are suffering from irritablesyndromes of the colon (irritable bowel syndrome or IBS), whileeffecting a complementary and simultaneous treatment of the symptomswhich are characteristic of the IBS.
 5. Use of pinaverium bromide forproducing a medicament which is intended for treating inflammatorydigestive diseases, such as inflammatory intestinal disease(inflammatory bowel disease or IBD), Crohn's disease or ulcerativecolitis, which diseases are generally associated with thickening of themucous membrane.
 6. Use according to claim 1 in which pinaverium bromideis used in sufficient quantity to be administered, by the oral route orby the injectable route, at the rate of from 50 to 450 mg per day usinga solution which is dosed at from 0.2 to 1 mg/ml.